Wide-spread dispersal in a deep-sea brooding polychaete: the role of natural history collections in assessing the distribution in quill worms (Onuphidae, Annelida).

BACKGROUND: Modern integrative taxonomy-based annelid species descriptions are detailed combining morphological data and, since the last decades, also molecular information. Historic species descriptions are often comparatively brief lacking such detail. Adoptions of species names from western literature in the past led to the assumption of cosmopolitan ranges for many species, which, in many cases, were later found to include cryptic or pseudocryptic lineages with subtle morphological differences. Natural history collections and databases can aid in assessing the geographic ranges of species but depend on correct species identification. Obtaining DNA sequence information from wet-collection museum specimens of marine annelids is often impeded by the use of  formaldehyde and/or long-term storage in ethanol resulting in DNA degradation and cross-linking. RESULTS: The application of ancient DNA extraction methodology in combination with single-stranded DNA library preparation and target gene capture resulted in successful sequencing of a 110-year-old collection specimen of quill worms. Furthermore, a 40-year-old specimen of quill worms was successfully sequenced using a standard extraction protocol for modern samples, PCR and Sanger sequencing. Our study presents the first molecular analysis of Hyalinoecia species including the previously known species Hyalinoecia robusta, H. tubicloa, H. artifex, and H. longibranchiata, and a potentially undescribed species from equatorial western Africa morphologically indistinguishable from H. tubicola. The study also investigates the distribution of these five Hyalinoecia species. Reassessing the distribution of H. robusta reveals a geographical range covering both the Atlantic and the Indian Oceans as indicated by molecular data obtained from recent and historical specimens. CONCLUSION: Our results represent an example of a very wide geographical distribution of a brooding deep-sea annelid with a complex reproduction strategy and seemingly very limited dispersal capacity of its offspring, and highlights the importance of molecular information from museum specimens for integrative annelid taxonomy and biogeography.

Head anatomy of a lantern shark wet-collection specimen (Chondrichthyes: Etmopteridae).

In this study, we apply a two-step (untreated and soft tissue stained) diffusible iodine-based contrast-enhanced micro-computed tomography array to a wet-collection Lantern Shark specimen of Etmopterus lucifer. The focus of our scanning approach is the head anatomy. The unstained CT data allow the imaging of mineralized (skeletal) tissue, while results for soft tissue were achieved after staining for 120 h in a 1% ethanolic iodine solution. Three-dimensional visualization after the segmentation of hard as well as soft tissue reveals new details of tissue organization and allows us to draw conclusions on the significance of organs in their function. Outstanding are the ampullae of Lorenzini for electroreception, which appear as the dominant sense along with the olfactory system. Corresponding brain areas of these sensory organs are significantly enlarged as well and likely reflect adaptations to the lantern sharks' deep-sea habitat. While electroreception supports the capture of living prey, the enlarged olfactory system can guide the scavenging of these opportunistic feeders. Compared to other approaches based on the manual dissection of similar species, CT scanning is superior in some but not all aspects. For example, fenestrae of the cranial nerves within the chondrocranium cannot be identified reflecting the limitations of the method, however, CT scanning is less invasive, and the staining is mostly reversible and can be rinsed out.

Cryptic persistence and loss of local endemism in Lake Constance charr subject to anthropogenic disturbance.

In the welcome circumstance that species believed extinct are rediscovered, it is often the case that biological knowledge acquired before the presumed extinction is limited. Efforts to address these knowledge gaps, in particular to assess the taxonomic integrity and conservation status of such species, can be hampered by a lack of genetic data and scarcity of samples in museum collections. Here, we present a proof-of-concept case study based on a multidisciplinary data evaluation approach to tackle such problems. The approach was developed after the rediscovery, 40 years after its presumed extinction, of the enigmatic Lake Constance deep-water charr Salvelinus profundus. Targeted surveys led to the capture of further species and additional sympatric normal charr, Salvelinus cf. umbla. Since the lake had been subject to massive stocking in the past, an evaluation of the genetic integrity of both extant forms was called for in order to assess possible introgression. A two-step genomic approach was developed based on restriction site associated DNA (RAD). Diagnostic population genomic (single nucleotide polymorphism [SNP]) data were harvested from contemporary samples and used for RNA bait design to perform target capture in DNA libraries of archival scale material, enabling a comparison between extant and historic samples. Furthermore, life history traits and morphological data for both extant forms were gathered and compared with historical data from the past 60-120 years. While extant deep-water charr matched historical deep-water specimens in body shape, gill raker count, and growth rates, significant differences were discovered between historical and extant normal charr. These resulted were supported by genomic analyses of contemporary samples, revealing the two extant forms to be highly divergent. The results of population assignment tests suggest that the endemic deep-water charr persisted in Lake Constance during the eutrophic phase, but not one of the historical genomic samples could be assigned to the extant normal charr taxon. Stocking with non-endemic charr seems to be the most likely reason for these changes. This proof-of-concept study presents a multidisciplinary data evaluation approach that simultaneously tests population genomic integrity and addresses some of the conservation issues arising from rediscovery of a species characterized by limited data availability.

Molecular phylogenetics of the Clupeiformes based on exon-capture data and a new classification of the order.

The Clupeiformes, including among others herrings, anchovies, shads and menhadens are ecologically and commercially important, yet their phylogenetic relationships are still controversial. Previous classification of Clupeiformes were based on morphological characters or lack of synapomorphic characters. More recent studies based on molecular data as well as new morphological evidence are keeping challenging their phylogenetic relations and there is still no consensus on many interrelationships within the Clupeiformes. In this study, we collected nuclear sequence data from 4,434 single-copy protein coding loci using a gene-capture method. We obtained a robust phylogeny based on 1,165 filtered loci with less than 30 % missing data. Our major findings include: 1) reconfirmation of monophyly of the Clupeiformes, that is, Denticipitidae is sister to all other clupeiforms; 2) the polyphyletic nature of dussumieriids and early branching of Spratelloididae from all other clupeoids were confirmed using datasets curated for less missing data and more balanced base composition in the respective taxa. The next branching clade is the monophyletic Engraulidae. Pristigasteridae also is monophyletic, but it was nested in the previously defined "Clupeidae". Within Pristigasteridae there is no support for monophyletic Pelloninae. Chirocentrus is close to Dussumieria and not to engraulids. The miniaturized Sundasalanx is placed close to the ehiravine Clupeonella, however, with a relatively deep split. The genus Clupea, is not part of the diverse "Clupeidae", but part of a clade containing additionally Sprattus and Etrumeus. Within the crown group clades, Alosidae and Dorosomatidae are retrieved as sister clades. Based on new fossil calibration points, we found that major lineages of the clupeiforms diverged in the late Cretaceous and early Paleogene. The extinction event at the end of the Cretaceous may have created ecological niches, which could have fueled the diversification of clupeiform fishes. Based on the strong evidence of the present study, we propose an updated classification of Clupeiformes consisting of ten families: Denticipitidae; Spratelloididae; Engraulidae (Engraulinae + Coiliinae); Clupeidae; Chirocentridae; Dussumieriidae; Pristigasteridae; Ehiravidae; Alosidae, Dorosomatidae.

A phylogenomic approach to reconstruct interrelationships of main clupeocephalan lineages with a critical discussion of morphological apomorphies.

BACKGROUND: Previous molecular studies on the phylogeny and classification of clupeocephalan fishes revealed numerous new taxonomic entities. For re-analysing these taxa, we perform target gene capturing and subsequent next generation sequencing of putative ortholog exons of major clupeocephalan lineages. Sequence information for the RNA bait design was derived from publicly available genomes of bony fishes. Newly acquired sequence data comprising > 800 exon sequences was subsequently used for phylogenetic reconstructions. RESULTS: Our results support monophyletic Otomorpha comprising Alepocephaliformes. Within Ostariophysi, Gonorynchiformes are sister to a clade comprising Cypriniformes, Characiformes, Siluriformes and Gymnotiformes, where the interrelationships of Characiformes, Siluriformes and Gymnotiformes remain enigmatic. Euteleosts comprise four major clades: Lepidogalaxiiformes, Protacanthopterygii, Stomiatii, and Galaxiiformes plus Neoteleostei. The monotypic Lepidogalaxiiformes form the sister-group to all remaining euteleosts. Protacanthopterygii, comprising Argentini-, Esoci- and Salmoniformes, is sister to Stomiatii (Osmeriformes and Stomiatiformes) and Galaxiiformes plus Neoteleostei. CONCLUSIONS: Several proposed monophyla defined by morphological apomorphies within the Clupeocephalan phylogeny are confirmed by the phylogenetic estimates presented herein. However, other morphologically described groups cannot be reconciled with molecular phylogenies. Thus, numerous morphological apomoprhies of supposed monophyla are called into question. The interpretation of suggested morphological synapomorphies of otomorph fishes is strongly affected by the inclusion of deep-sea inhabiting, and to that effect morphologically adapted Alepocephaliformes. Our revision of these potential synapomorphies, in the context that Alepocephaliformes are otomorph fishes, reveals that only a single character of the total nine characters proposed as synapomorphic for the group is clearly valid for all otomorphs. Three further characters remain possible apomorphies since their status remains unclear in the deep-sea adapted Alepocephaliformes showing developmental lag and lacking a swim bladder. Further, our analysis places Galaxiiformes as sister group to neoteleosts, which contradicts some previous molecular phylogenetic studies. This needs further investigation from a morphological perspective, as suggested synapomophies for several euteleostean lineages are challenged or still lacking. For the verification of results presented herein, a denser phylogenomic-level taxon sampling should be applied.

DNA capture reveals transoceanic gene flow in endangered river sharks.

For over a hundred years, the "river sharks" of the genus Glyphis were only known from the type specimens of species that had been collected in the 19th century. They were widely considered extinct until populations of Glyphis-like sharks were rediscovered in remote regions of Borneo and Northern Australia at the end of the 20th century. However, the genetic affinities between the newly discovered Glyphis-like populations and the poorly preserved, original museum-type specimens have never been established. Here, we present the first (to our knowledge) fully resolved, complete phylogeny of Glyphis that includes both archival-type specimens and modern material. We used a sensitive DNA hybridization capture method to obtain complete mitochondrial genomes from all of our samples and show that three of the five described river shark species are probably conspecific and widely distributed in Southeast Asia. Furthermore we show that there has been recent gene flow between locations that are separated by large oceanic expanses. Our data strongly suggest marine dispersal in these species, overturning the widely held notion that river sharks are restricted to freshwater. It seems that species in the genus Glyphis are euryhaline with an ecology similar to the bull shark, in which adult individuals live in the ocean while the young grow up in river habitats with reduced predation pressure. Finally, we discovered a previously unidentified species within the genus Glyphis that is deeply divergent from all other lineages, underscoring the current lack of knowledge about the biodiversity and ecology of these mysterious sharks.

Molecular phylogeny of Squaliformes and first occurrence of bioluminescence in sharks.

BACKGROUND: Squaliform sharks represent approximately 27 % of extant shark diversity, comprising more than 130 species with a predominantly deep-dwelling lifestyle. Many Squaliform species are highly specialized, including some that are bioluminescent, a character that is reported exclusively from Squaliform sharks within Chondrichthyes. The interfamiliar relationships within the order are still not satisfactorily resolved. Herein we estimate the phylogenetic interrelationships of a generic level sampling of "squaloid" sharks and closely related taxa using aligned sequences derived from a targeted gene capture approach. The resulting phylogenetic estimate is further used to evaluate the age of first occurrence of bioluminescence in Squaliformes. RESULTS: Our dataset comprised 172 putative ortholog exon sequences. Phylogenetic estimates result in a fully resolved tree supporting a monophyletic lineage of Squaliformes excluding Echinorhinus. Non-luminous Squalidae are inferred to be the sister to a clade comprising all remaining Squaliform families. Our results suggest that the origin of photophores is coincident with an elevated diversification rate and the splitting of families Dalatiidae, Etmopteridae, Oxynotidae and Somniosidae at the transition of the Lower to the Upper Cretaceous. The presence of luminous organs was confirmed for the Sleeper shark genus Zameus. These results indicate that bioluminescence in sharks is not restricted solely to the families Etmopteridae and Dalatiidae as previously believed. CONCLUSIONS: The sister-clade to non-luminous Squalidae comprises five families. The presence of photophores is reported for extant members of three out of these five families based on results of this study, i.e. Lantern sharks (Etmopteridae), Kitefin sharks (Dalatiidae) and Sleeper sharks (Somniosidae). Our results suggest that the origin of luminous organs arose during the rapid diversification event that gave rise to the extant Squaliform families. These inferences are consistent with the idea of diversification of Squaliform sharks being associated with the emergence of new deep-sea habitats in the Lower Cretaceous, which may have been facilitated by the evolution of bioluminescence.

Shark genome size evolution and its relationship with cellular, life-history, ecological, and diversity traits.

Among vertebrates, sharks exhibit both large and heterogeneous genome sizes ranging from 2.86 to 17.05 pg. Aiming for a better understanding of the patterns and causalities of shark genome size evolution, we applied phylogenetic comparative methods to published genome-size estimates for 71 species representing the main phylogenetic lineages, life-histories and ecological traits. The sixfold range of genome size variation was strongly traceable throughout the phylogeny, with a major expansion preceding shark diversification during the late Paleozoic and an ancestral state (6.33 pg) close to the present-day average (6.72 pg). Subsequent deviations from this average occurred at higher rates in squalomorph than in galeomorph sharks and were unconnected to evolutionary changes in the karyotype architecture, which were dominated by descending disploidy events. Genome size was positively correlated with cell and nucleus sizes and negatively with metabolic rate. The metabolic constraints on increasing genome size also manifested at higher phenotypic scales, with large genomes associated with slow lifestyles and purely marine waters. Moreover, large genome sizes were also linked to non-placental reproductive modes, which may entail metabolically less demanding embryological developments. Contrary to ray-finned fishes, large genome size was associated neither with the taxonomic diversity of affected clades nor with low genetic diversity.

A DNA sequence-based identification checklist for Taiwanese chondrichthyans.

In an effort to establish a DNA sequence based checklist of the highly diverse chondrichthyan fauna of Taiwan, we sequenced the mitochondrial NADH2 gene of 257 freshly sampled specimens of Taiwanese chondrichthyans, which were identified to species level by experts in the field. The newly generated sequences were analysed in the context of an already published phylogeny based on NADH2 sequences of chondrichthyans to identify taxonomically interesting lineages as well as enhancing the already published phylogeny with species hitherto not included. Results show a number of noteworthy findings indicating distinct population differences or cryptic diversity and enhance published data with 22 new species not analyzed so far. Our study may be helpful for further taxonomic work on the Taiwanese chrondrichthyan fauna.

An annotated checklist of the chondrichthyans of Taiwan.

An annotated checklist of chondrichthyan fishes (sharks, batoids, and chimaeras) occurring in Taiwanese waters is presented. The checklist is the result of a biodiversity workshop held in Mach 2012 as well as on-going systematic revisions by the authors. The chondrichthyan fauna of Taiwan is one of the richest in the world with the number of species totaling 181, comprising 52 families and 98 genera. It includes 31 families, 64 genera, and 119 species of sharks, 19 families, 31 genera, and 58 species of batoids, and 2 families, 3 genera, and 4 species of chimaeras. The most species-rich families are the Carcharhinidae with 22 species followed by the Scyliorhinidae with 17. The most species-rich batoid families are the Dasyatidae with 11 species and and the Rajidae with 10. Verified voucher material is provided for each species where available and potential taxonomic issues are high-lighted when applicable. This represents the first detailed, evidence-based checklist of chondrichthyans from Taiwanese waters in over 40 years.

Molecular identification and first demographic insights of sharks based on artisanal fisheries bycatch in the Pacific Coast of Colombia: implications for conservation.

The Pacific coast of Colombia is characterized by mangrove ecosystems which play a crucial role as possible nurseries for juvenile sharks. However, trophic food webs from coastal ecosystems are heavily disturbed by increased fishing pressure, which affects numerous shark species. In this region of the Eastern Tropical Pacific (ETP), fisheries' data from coastal areas are scarce and unspecific, as most sharks from artisanal fisheries are landed decapitated and finless, making their morphological identification difficult. For the establishment and implementation of effective regional conservation and management policies, information on the diversity and population dynamics of shark species is crucial. We therefore sequenced the mitochondrial NADH2 gene of 696 samples taken from fishermen's landings of shark's bycatch along the Colombian north Pacific coast. We were able to identify 14 species of sharks, two of the most abundant species were Sphyrna lewini and Carcharhinus falciformis, both evaluated on IUCN the Red List of Threatened species (Critically Endangered and Vulnerable) and CITES regulated. We found low genetic diversity in the sampled area increasing the concern for both species in the region, even more considering that the majority of individuals were juveniles. Our results showed the importance of genetic markers for first population genetic insights as a complementary tool during the decision-making process in management plans. For this specific region, strategies such as the delimitation of conservation priority areas or the regulation of fishing gears could help improve the sustainability of shark populations in the Colombian Pacific.

Trophic position of Otodus megalodon and great white sharks through time revealed by zinc isotopes.

Diet is a crucial trait of an animal's lifestyle and ecology. The trophic level of an organism indicates its functional position within an ecosystem and holds significance for its ecology and evolution. Here, we demonstrate the use of zinc isotopes (δ66Zn) to geochemically assess the trophic level in diverse extant and extinct sharks, including the Neogene megatooth shark (Otodus megalodon) and the great white shark (Carcharodon carcharias). We reveal that dietary δ66Zn signatures are preserved in fossil shark tooth enameloid over deep geologic time and are robust recorders of each species' trophic level. We observe significant δ66Zn differences among the Otodus and Carcharodon populations implying dietary shifts throughout the Neogene in both genera. Notably, Early Pliocene sympatric C. carcharias and O. megalodon appear to have occupied a similar mean trophic level, a finding that may hold clues to the extinction of the gigantic Neogene megatooth shark.

Mitochondrial DNA sequencing of a wet-collection syntype demonstrates the importance of type material as genetic resource for lantern shark taxonomy (Chondrichthyes: Etmopteridae).

After initial detection of target archival DNA of a 116-year-old syntype specimen of the smooth lantern shark, Etmopterus pusillus, in a single-stranded DNA library, we shotgun-sequenced additional 9 million reads from this same DNA library. Sequencing reads were used for extracting mitochondrial sequence information for analyses of mitochondrial DNA characteristics and reconstruction of the mitochondrial genome. The archival DNA is highly fragmented. A total of 4599 mitochondrial reads were available for the genome reconstruction using an iterative mapping approach. The resulting genome sequence has 12 times coverage and a length of 16 741 bp. All 37 vertebrate mitochondrial loci plus the control region were identified and annotated. The mitochondrial NADH2 gene was subsequently used to place the syntype haplotype in a network comprising multiple E. pusillus samples from various distant localities as well as sequences from a morphological similar species, the shortfin smooth lantern shark Etmopterus joungi. Results confirm the almost global distribution of E. pusillus and suggest E. joungi to be a junior synonym of E. pusillus. As mitochondrial DNA often represents the only available reference information in non-model organisms, this study illustrates the importance of mitochondrial DNA from an aged, wet collection type specimen for taxonomy.

Successful application of ancient DNA extraction and library construction protocols to museum wet collection specimens.

Millions of scientific specimens are housed in museum collections, a large part of which are fluid preserved. The use of formaldehyde as fixative and subsequent storage in ethanol is especially common in ichthyology and herpetology. This type of preservation damages DNA and reduces the chance of successful retrieval of genetic data. We applied ancient DNA extraction and single stranded library construction protocols to a variety of vertebrate samples obtained from wet collections and of different ages. Our results show that almost all samples tested yielded endogenous DNA. Archival DNA extraction was successful across different tissue types as well as using small amounts of tissue. Conversion of archival DNA fragments into single-stranded libraries resulted in usable data even for samples with initially undetectable DNA amounts. Subsequent target capture approaches for mitochondrial DNA using homemade baits on a subset of 30 samples resulted in almost complete mitochondrial genome sequences in several instances. Thus, application of ancient DNA methodology makes wet collection specimens, including type material as well as rare, old or extinct species, accessible for genetic and genomic analyses. Our results, accompanied by detailed step-by-step protocols, are a large step forward to open the DNA archive of museum wet collections for scientific studies.

Molecular phylogeny and node time estimation of bioluminescent Lantern Sharks (Elasmobranchii: Etmopteridae).

Deep-sea Lantern Sharks (Etmopteridae) represent the most speciose family within Dogfish Sharks (Squaliformes). We compiled an extensive DNA dataset to estimate the first molecular phylogeny of the family and to provide node age estimates for the origin and diversification for this enigmatic group. Phylogenetic inferences yielded consistent and well supported hypotheses based on 4685bp of both nuclear (RAG1) and mitochondrial genes (COI, 12S-partial 16S, tRNAVal and tRNAPhe). The monophyletic family Etmopteridae originated in the early Paleocene around the C/T boundary, and split further into four morphologically distinct lineages supporting three of the four extant genera. The exception is Etmopterus which is paraphyletic with respect to Miroscyllium. Subsequent rapid radiation within Etmopterus in the Oligocene/early Miocene was accompanied by divergent evolution of bioluminescent flank markings which morphologically characterize the four lineages. Higher squaliform interrelationships could not be satisfactorily identified, but convergent evolution of bioluminescence in Dalatiidae and Etmopteridae is supported.

The presence of lateral photophores correlates with increased speciation in deep-sea bioluminescent sharks.

The vast majority of species within the lanternshark genus Etmopterus harbour complex luminescent markings on their flanks, whose functional significance has long remained obscure. Recent studies, however, suggest these enigmatic photophore aggregations to play a role in intraspecific communication. Using visual modelling based on in vivo luminescence measurements from a common lanternshark species, we show that etmopterid flank markings can potentially work as a medium range signal for intraspecific detection/recognition. In addition, using molecular phylogenetic analyses, we demonstrate that the Etmopterus clade exhibits a greater than expected species richness given its age. This is not the case for other bioluminescent shark clades with no (or only few) species with flank markings. Our results therefore suggest that etmopterid flank markings may provide a way for reproductive isolation and hence may have facilitated speciation in the deep-sea.

Iso-luminance counterillumination drove bioluminescent shark radiation.

Counterilluminating animals use ventral photogenic organs (photophores) to mimic the residual downwelling light and cloak their silhouette from upward-looking predators. To cope with variable conditions of pelagic light environments they typically adjust their luminescence intensity. Here, we found evidence that bioluminescent sharks instead emit a constant light output and move up and down in the water column to remain cryptic at iso-luminance depth. We observed, across 21 globally distributed shark species, a correlation between capture depth and the proportion of a ventral area occupied by photophores. This information further allowed us, using visual modelling, to provide an adaptive explanation for shark photophore pattern diversity: in species facing moderate predation risk from below, counterilluminating photophores were partially co-opted for bioluminescent signalling, leading to complex patterns. In addition to increase our understanding of pelagic ecosystems our study emphasizes the importance of bioluminescence as a speciation driver.

Capturing protein-coding genes across highly divergent species.

DNA hybridization capture combined with next generation sequencing can be used to determine the sequences of hundreds of target genes across hundreds of individuals in a single experiment. However, the approach has thus far only been successfully applied to capture targets that are highly similar in sequence to the bait molecules. Here we introduce modifications that extend the reach of the method to allow efficient capture of highly divergent homologous target sequences using a single set of baits. These modifications have important implications for comparative biology.